This invention relates to instrumentation for fluorometric analysis of polymerase chain reaction (PCR) samples wherein the amplified product is bound to an intercalating fluorescent dye and more particularly to a holder which provides rapid and convenient manual progression of reaction strips used as cuvettes in a fluorometer.
Polymerase chain reaction ("PCR") is a powerful analytical tool which has become increasingly popular and useful in a variety of applications, from matching bodily fluid samples to a defendant in criminal procedures to identifying unknown organisms in a biological or environmental sample. PCR is an assay which amplifies a desired specific nucleic acid fragment or sequence contained in a nucleic acid or mixture of nucleic acids. The PCR assay is based on a protocol of thermal incubation in which the sample is subjected to a repeated sequence of alternating elevated temperatures for controlled time periods. Higher and lower temperature incubations are alternated in a repeated sequence or cycle, and the cycle is then repeated for a specified number of times.
Following PCR, existence of an amplified product must be determined. There are a number of known methods. The most common involves gel electrophoresis in which wells associated with individual lanes are charged by pipette with a sample or a control. An electrical potential is applied across the wells causing DNA fragments to migrate at rates proportional to size and electrical charge. After separation, the gel is stained as with ethidium bromide, which fluoresces when properly excited, and read.
Another method involves capturing or immobilizing reaction products in a 96-well microtiter plate, commercially available from ICN Titertek and read on a commercial plate reader such as the Fluoroskan, also available from ICN Titertek. A modified 96-well plate with tapered cavities ("V-bottom") specifically sized for the tubes used in the Perkin Elmer cycler is available from Evergreen. This fits into a plate reader such as the Fluoroskan.
Still another technique, disclosed in Canadian Patent Application 2,067,909, uses a DNA binding agent which provides a detectable signal when bound to double-stranded nucleic acid which signal is distinguishable from the signal when it is unbound. The technique employs an intercalating agent, such as a fluorescent dye (e.g., ethidium bromide) and a spectra fluorometer in conjunction with optical fibers.
These above procedures and techniques have several disadvantages. First, any technique which requires the sample to be transferred, such as by pipette, to another container or surface presents the possibility of contamination. In addition, samples must be prepared in a isolated room or hood to avoid contamination from the room where the tubes are opened. Accordingly, it is preferred that the sample be "read" in its original container. Second, the large 96-well plates, while adequate for large numbers of samples, are often cumbersome and unwieldy when only a few samples need to be read. Third, reading of multiple well plates must be done from the top, removing the source of the signal from the sensor. Fourth, electrophoretic gels can be difficult to produce, handle and develop and suffer from several of the problems related above as well.